The present invention relates in part with the observations that the cholesterol epoxides are found in animal products arising either metabolically or by autoxidation. In the presence of molecular oxygen and light cholesterol readily autoxidizes to form predominantly cholesterol 5.beta.,6.beta.-epoxide and a minor proportion of the isomeric, cholesterol 5.beta.,6.beta.-epoxide. The degree of autoxidation increases with time and temperature. The cholesterol epoxides, thus, are found in aged cholesterol-rich products, such as dried egg products.
Of major importance, however, are the cholesterol epoxides which form metabolically in a variety of organs and tissues, such as the liver, male prostate gland, and female breast. As highly transient metabolic intermediates in the pathway from cholesterol to cholestane 3.beta.,5.alpha.,6.beta.-triol, a suspected regulator of endogenous cholesterol synthesis, the cholesterol epoxides normally never accumulate nor are they generally detectable with analytical procedures currently available to clinical scientists.
However, associated with various pre-developmental or existing pathological states, the cholesterol epoxides do accumulate in the related tissues and secretions. Initially, as a dose-dependent response cholesterol 5.alpha.,6.alpha.-epoxide was detected in the skin of humans and animals after exposure to ultraviolet irradiation. Likewise the cholesterol epoxides are present in the serum of patients suffering with familial hypercholesterolemia where the serum cholesterol levels are also elevated. Tissue epoxycholesterol accumulation has also been seen in the rare but fatal Wolman's disease.
Of major importance is the more recent observation that the cholesterol epoxides are found in the tissues and secretions of the aging human prostate gland. This observation is particularly consistent with the development of benign and malignant diseases. The epoxycholesterols have also been observed with aging in female breast secretions and are associated with the high risk category for development of breast cancer. The female breast and male prostate are both hormone-regulated glandular secretory organs producing significant quantities of cholesterol. Significant increases of the cholesterol content of serum, prostate tissues and breast aspirates are now associated with the appearance of the epoxycholesterols, suggesting some loss of regulation of cholesterol synthesis and metabolism.
The possible role of cholesterol and its metabolites in mutagenesis and carcinogenesis has long been a controversial subject. The earliest observation revealed the production of sarcomas and other tumors when cholesterol epoxides were administered subcutaneously to experimental animals. The formation of tumors after ultraviolet irradiation of skin has been correlated with the initial formation of cholesterol 5.alpha.,6.alpha.-epoxide. Likewise, cholesterol epoxides, due to the angiotoxicity of oxygenated sterols and not cholesterol itself, are suspect in the development of arterial wall damage leading to the eventual emergence of atherosclerotic lesions and cardiovascular blockage.
There is sufficient evidence that the cholesterol epoxides produce a similar degree of chromosome damage and DNA repair synthesis as low doses of ultraviolet irradiation. Cholesterol 5.alpha.,6.alpha.-epoxide forms a strong physical bond with DNA leading to significant covalent attachment of the steroid to this macromolecule. Likewise, it has been demonstrated with hamster embryonic cells that cholesterol 5.alpha.,6.alpha.-epoxide is as potent a carcinogen as the well recognized 3-methylcholanthrene. This cholesterol metabolite has also been implicated as an etiologic agent in human colon cancer.
In considering that many well known exogenous carcinogens undergo metabolic activation to the electrophilic "ultimate carcinogen" by endogeneous metabolic epoxidation reactions, it would be expected that the electrophilic cholesterol epoxides may also play an important role in a wide variety of cytotoxic, mutagenic and carcinogenic physiological reactions. Their detection and quantitation in biological fluids and tissues has become of increasing importance in clinical medicine.
The consistent presence of the epoxycholesterols in prostatic secretions may be diagnostic for the development of both benign and malignant diseases of the human and canine prostate gland. Likewise, detection of the cholesterol epoxides in female breast aspirates may be associated as a risk factor for the development of benign breast disease and breast cancer. Since the cholesterol epoxides are detected in the serum of patients suffering with familial hypercholesterolemia which normally results in early death due to advanced atherosclerosis, the appearance of these cholesterol metabolites in serum may serve as an important risk factor for the development of coronary vascular disease.
The qualitative and quantitative measurement of the cholesterol epoxides in biological fluids and tissues due to their relatively low concentration has been a difficult, costly and time-consuming task. The procedures of thin layer chromatography, high performance liquid chromatography, gas-liquid chromatography, nuclear magnetic resonance spectrometry, and mass spectrometry have all been employed either alone or in combination for the detection, characterization and quantitative measurement of the cholesterol epoxides in different biological fluids and tissues. While important in research projects as experimental procedures, these do not lend themselves readily to routine, quick, precise and economical clinical analyses in medical practice.
A usual procedure for the development of the immunoassay for cholesterol epoxides might follow a series of well-established methods. Normally, cholesterol epoxide, itself not antigenic as a "hapten", would be complexed through stable covalent bonds to a normally antigenic molecule such as a protein. Protein "carriers", such as bovine serum albumin, ovalbumin and bovine gamma globulin are often employed for this purpose. The cholesterol epoxide-protein complex or "immunogen" would then be introduced to the blood of some living experimental animal such as the mouse, rat or rabbit. With the recognition of the presence of a foreign antigenic substance, the animal in turn would then consequently produce a specific protein called "antibody" which has the specific ability to complex with the foreign cholesterol epoxide-containing protein or with cholesterol epoxide itself. The production of this specific antibody by the animal is the essential step in the development of an immunoassay test procedure for cholesterol epoxide. The isolation of this specific antibody protein from the blood of the animal would enable the preparation of one essential component or reagent of the immunoassay test procedure.
The reaction of the cholesterol epoxide specific antibody protein with the test product whether in serum or in breast or prostatic secretions would result in the formation of complexes that can be isolated. If the test product, cholesterol epoxide, is labelled with some enzyme or radioactive element, the amount of the label in the resultant complex then depends on the amount of product that was added for a fixed amount of standardized reagent antibody. If one combines labelled test product, as a known reference, with an unknown sample from a test patient, the product in the test sample will compete with the labelled reference product in reacting with the antibody. This will result in a decrease in the amount of label combined with the antibody. This decrease usually provides a sensitive and precise measure of the product, cholesterol epoxide, in the test sample of the patient. Thus, the cholesterol epoxide specific antibody protein and the enzyme--or radioactive--labelled cholesterol epoxide test product are the essential components of an enzyme-immuno or radioimmuno assay test procedure, respectively.
Immunoassay test procedures whether based on enzyme or radioactive element-linked antibody assays are ordinarily extremely sensitive, highly specific, and rapid by nature. While such procedures are not usually employed for the quantitation of cholesterol, itself, they are currently widely employed for the clinical analysis of other cholesterol-derived molecules, such as the steroid hormones, testosterone, 5.alpha.-dehydrotestosterone, estradiol, estrone, estriol-17.beta., cortisol and cortisone which like the cholesterol epoxides are normally only present in small quantities in biological fluid and tissues.
However, there is a complication encountered in the immunoassay of particular steroid hormones in that there is usually cross-reactivity of other steroids of related or similar molecular structure. As an example, antiserum for estradiol-17.beta. could exhibit percentage cross-reaction of 94, 61, and 19 for estrone, estradiol-17.alpha., and estriol-17.beta., respectively. In considering the concentration of the cholesterol epoxides in biological fluids such as serum and breast or prostatic secretions, one always finds a most significant excess of cholesterol as well. As much as a 40-100 fold greater concentration of cholesterol is usually found as compared to that of the cholesterol epoxides. Thus, with the preparation of antibodies directed towards the intact cholesterol epoxide-protein immunogen, it would be expected that cholesterol itself would exhibit some cross-reactivity with this antibody. This would reduce the usefulness of the immunoassay procedure for the cholesterol epoxides by virtue of the possibility that cholesterol itself would react, leading to false positive results.
It is therefore an object of the present invention to provide a clinical diagnostic method for the qualitative and quantitative measurement of the cholesterol epoxides based on immunological or immunoassay procedures.